2.1 Interferon Stimulated Gene ISG15
The type I interferon (IFN) pathway plays an essential role in anti-viral immunity by inducing the expression of hundreds of genes. Some interferon stimulated genes (ISG) have well-characterized anti-viral functions, but for the majority of ISGs, their mechanism of action is unknown. ISG15 was first identified as a 15 kDa protein induced by IFN treatment that cross-reacted with anti-ubiquitin antibodies (Blomstrom et al., 1986; Haas et al., 1987; Korant et al., 1984). A member of the ubiquitin-like (Ubl) family, ISG15 possesses significant homology to a di-ubiquitin sequence. The crystal structure of ISG15 revealed two ubiquitin folds joined by a linker sequence (Narasimhan et al., 2005). Like ubiquitin, ISG15 is linked to target proteins via an isopeptide bond between the terminal carboxyl group of ISG15 and the ε-amino group of lysines of target proteins (Ritchie and Zhang, 2004; Welchman et al., 2005).
ISG15 utilizes a series of IFN-induced enzymes to conjugate to target proteins. The activating enzyme (E1) and the conjugating enzyme (E2) have been identified as UBE1L (Yuan and Krug, 2001) and UbcH8 (Zhao et al., 2004), respectively. More recently, Herc5 (Dastur et al., 2006) and EFP (Zou and Zhang, 2006) have been found to be ISG15 ligases (E3). DeISGylation is catalyzed by UBP43, an ISG15-specific deconjugating enzyme (Malakhov et al., 2002). To date, the only ISG15-specific isopeptidase that had been identified is UBP43. No viral protein has been demonstrated to have deISGylating activity, however it has been demonstrated that the de-ubiquitinating proteases Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) PLpro and the adenovirus protease can process ISG15 fusion proteins (Balakirev et al., 2002; Barretto et al., 2005; Lindner et al., 2005). Proteomic approaches have identified target proteins for ISGylation (Giannakopoulos et al., 2005; Zhao et al., 2005). Some of these proteins, such as STAT-1, PKR, Mx and RIG-I, are also IFNα/β-inducible and have known anti-viral functions (Haller et al., 1995; Meurs et al., 1992; Yoneyama et al., 2004). The existence of an IFN-inducible pathway that mirrors ubiquitin conjugation suggests that protein ISGylation may be an important component of the IFN-induced innate immune response.
2.2 Anti-Viral Properties of ISG15
Numerous studies have suggested that ISG15 has anti-viral properties in vivo. When ISG15 was heterologously expressed from a recombinant Sindbis virus, the Sindbis virus was no longer lethal to IFNαβR1−/− mice (Lenschow et al., 2005). ISG15−/− mice display increased susceptibility to infection with Sindbis, herpes simplex, influenza A and influenza B viruses (Lenschow et al., 2007), although they have no defect in the antiviral response against vesicular stomatitis virus (VSV) or lymphocytic choriomeningitis virus (LCMV) (Osiak et al., 2005). ISGylation has been reported to inhibit HIV budding (Okumura et al., 2006). In addition, the NS1 of influenza B virus specifically binds ISG15 and blocks cellular protein ISGylation by inhibiting the ISG15-UBE1L interaction, most likely as an immune evasion mechanism (Yuan et al., 2002; Yuan and Krug, 2001).
2.3 Ovarian-Tumor (OTU) Domain-Containing Proteins of the Deubiquitinating (DUB) Superfamily
Ubiquitin (Ub) conjugation and deconjugation have been shown to play an essential role in the regulation of numerous biological processes, including protein degradation, signal transduction and endocytosis. The deubiquitinating (DUB) superfamily of proteases includes more than 500 members, most of them specific for Ub, and only a few of them displaying activity towards Ubl molecules (Nyman et al., 2000). Some mammalian proteins (such as Otubains, Cezanne, and A20) belonging to the Ovarian Tumor (OTU) family of cysteine proteases have been described as a DUB (Balakirev et al., 2003; Evans et al., 2004; Evans et al., 2003).
2.4 Crimean Congo Hemorrhagic Fever Virus (CCHFV)
Crimean Congo Hemorrhagic Fever Virus (CCHFV) is a human pathogen distributed mainly in Africa, Asia and Eastern Europe. It is the causative agent for a tick-borne hemorrhagic fever with high mortality rates (Flick and Whitehouse, 2005). CCHFV is a segmented negative-stranded RNA virus belonging to the nairovirus genus in the bunyaviridae family. Nairoviruses differ from other bunyaviruses in the relative sizes of their tri-segmented genome and particularly in the large L segment that encodes a putative RNA dependent RNA polymerase (RdRp) of 450 kDa.